The interactions of cells with the extracellular matrix (ECM) are critical for the normal development and function of the organism. Modulation of cell-matrix interactions occurs through the action of unique proteolytic systems responsible for hydrolysis of a variety of ECM components. By regulating the integrity and composition of the ECM structure, these enzyme systems play a pivotal role in the control of signals elicited by matrix molecules, which regulate cell proliferation, differentiation, and cell death. The turnover and remodeling of ECM must be highly regulated since uncontrolled proteolysis contributes to abnormal development and to the generation of many pathological conditions characterized by either excessive degradation or a lack of degradation of ECM components.
Matrix metalloproteinases (MMPs) are a major group of enzymes that regulate cell-matrix composition. The MMPs are zinc-dependent endopeptidases known for their ability to cleave one or several ECM constituents as well as nonmatrix proteins. They comprise a large family of proteases that share common structural and functional elements and are products of different genes. Ample evidence exists on the role of MMPs in normal and pathological processes, including embryogenesis, wound healing, inflammation, arthritis, apoptosis and cancer. The association of MMPs with cancer metastasis has raised considerable interest because they represent an attractive target for development of novel antimetastatic drugs aimed at inhibiting MMP activity. Therefore, understanding the structure and function of these key enzymes has significant implications for cancer therapy. Massova, I., et al., (1998) The FASEB Journal, 12:1075-1095.
Most members of the MMP family are organized into three basic, distinctive, and well-conserved domains based on structural considerations: an amino-terminal propeptide; a catalytic domain; and a hemopexin-like domain at the carboxy-terminal. The propeptide consists of approximately 80-90 amino acids containing a cysteine residue, which interacts with the catalytic zinc atom via its side chain thiol group. A highly conserved sequence (. . . PGCGXPD . . . ) is present in the propeptide. Removal of the propeptide by proteolysis results in zymogen activation, as all members of the MMP family are produced in a latent form. The catalytic domain contains two zinc ions and at least one calcium ion coordinated to various residues. One of the two zinc ions is present in the active site and is involved in the catalytic processes of the MMPs. The second zinc ion (also known as structural zinc) and the calcium ion are present in the catalytic domain approximately 12 .ANG. away from the catalytic zinc. The catalytic zinc ion is essential for the proteolytic activity of MMPs; the three histidine residues that coordinate with the catalytic zinc are conserved among all the MMPs. Little is known about the roles of the second zinc ion and the calcium ion within the catalytic domain, but the MMPs are shown to possess high affinities for structural zinc and calcium ions. Bode, W., et al., (1994) EMBO Journal, 13:1263-1269; Salowe S. P., et al., (1992) Biochemistry 31:4535-4540. The hemopexin-like domain of MMPs is highly conserved and shows sequence similarity to the plasma protein, hemopexin. The hemopexin-like domain has been shown to play a functional role in substrate binding and/or in interactions with the tissue inhibitors of metalloproteinases (TIMPs), a family of specific MMP protein inhibitors. Borden, P., and Heller R. A. (1997) Crit. Rev. Eukaryot. Gen. Expression 7:159-178; Gomis-Ruth, F. X., et al., Nature (London) 389:77-81. In addition to these basic domains, the family of MMPs evolved into different subgroups by incorporating and/or deleting structural and functional domains.
Neutrophil collagenase (MMP-8) is a member of the matrix metalloproteinase (MMP) family. It is capable of cleaving all three .alpha.-chains of types I, II, and III collagen. It is a secreted glycoprotein which is synthesized as a latent enzyme. The nucleotide sequence of MMP-8 cDNA (GenBank Accession No. J05556) encodes a protein of 467 amino acids, with a secretory signal sequence of 20 residues followed by the prodomain of 80 residues. The activation of this enzyme requires autolytic removal of 80 amino acids from the N-terminus. Devarajan, P., et al., (1991) Blood 77, 2731-2738; Hasty, K.A., et al., (1990) J. Biol. Chem. 265, 11421-11424. MMP-8 was previously thought to be expressed exclusively by neutrophils, but recently its expression has also been detected in chondrocytes and was found to be capable of cleaving aggrecan in cartilage. Amer, E.C., et al., (1997) J. Biol. Chem. 272, 9294-9299; Cole, A.A. et al. (1996) J. Biol. Chem. 271, 11023-11026; and Cole, A.A. and Kuettner, K.E. (1995) Acta Orthop. Scand. Suppl. 266, 98-102.
Recent studies have shown that MMP-8 is not a unique gene product of neutrophils since it is also expressed by chondrocytes in human articular cartilage Cole, A.A. et al. (1996) J. Biol. Chem. 271, 11023-11026; and Cole, A.A. and Kuettner, K.E. (1995) Acta Orthop. Scand. Suppl. 266, 98-102. It is capable of cleaving not only collagen but also aggrecan. Further, MMP-8 mRNA expression has been observed in mononuclear fibroblast-like cells in the rheumatoid synovial membrane and Western blot analysis shows a similar up-regulation at the protein level in arthritic conditions. Hanemaaijer R., et al., (1997) J. Biol. Chem. 12:272(50):31504-9. Accordingly, MMP-8 is involved in the causation of arthritis.
Pre-mRNA splicing is a widely used biological mechanism in higher eukaryotes for generating mature mRNA. More recently, it has become apparent that pre-mRNAs of many genes are capable of undergoing alternative splicing and generate multiple species of mature mRNA. Some of the splice variants occur at the non-coding region of the mRNA and do not influence the amino acid sequence of the translation products but may somehow affect translation efficiency. Rescheleit, D.K., et al., (1996) FEBS Letters 394, 345-348. On the other hand, alternative splicing can also occur at the coding region of the mRNA, resulting in translation products with different tissue distribution or subcellular localization. Kato, A., et al., (1997) J. Biol. Chem. 272, 15313-15322; Joun, H., et al., (1997) Endocrinology 138, 1742-1749; and Nilsen, H., et al., (1997) Nucleic Acids Res. 25, 750-755. The ability to detect such splicing variants for a given mRNA has greatly increased due to the use of polymerase chain reaction (PCR) coupled with the reverse transcription (RT) of mRNA.
Several members of MMP family whose genomic structure have been analyzed all contain an intron at the similar position as MMP-8. Collier, I.E., et al., (1988) J. Biol. Chem. 263, 10711-10713; Anglard, P., et al., (1995) J. Biol. Chem. 270, 20337-20344; and Pendas, A.M., et al., (1997) Genomics 40, 222-233. These include both collagenase-1 (MMP-1) and collagenase-3 (MMP-13). In the case of membrane-type MMPs, an extended family of MMP, an alternatively spliced MT-MMP-3 was identified recently. Matsumoto, S., et al. (1997) Biochim Biophys Acta 1354, 159-170. This alternative splicing occurs near the transmembrane region of MT-MMP-3, which results in soluble instead of membrane-anchored MT-MMP-3. RT-PCR has made it possible to identify alternative spliced form of MMPs.
In addition to extracellular matrix, the cytoskeleton proteins inside of cells are also essential for cellular functions such as vesicle movement inside of cells, cell division and migration, and even cell survival. Consequently, excess degradation of cytoskeleton proteins may also lead to arthritis, cancer, and disease caused by cellular apoptosis including but not limited to Parkinson's disease, Alzheimer's disease and Huntington's chorea. Therefore, there is a need for a process to diagnose the onset and progression of cytoskeleton protein degradation in order to assess appropriate therapeutic measures and their effectiveness.
There is also a need to detect and measure the differential expression of genes and gene products which are altered in this disease state, such that this differential expression can be determined diagnostically to predict the onset of the disease state.
A further need exists for identifying additional factor(s) and others which interact with and regulate the biological function of the gene and gene products which show differential expression in cytoskeleton protein degradation, so that they may be administered to patients in need of such treatment.
There also exists a need for pharmaceuticals comprising the factor(s) which interact with the gene and gene products that are active in the above-described disease states and/or are differentially expressed in these disease states such that they may be administered to a patient in need thereof for the treatment and/or prevention of these disease states.